Genetic and Pathogenic Characterization of Avian Influenza Virus in Migratory Birds between 2015 and 2019 in Central China

ABSTRACT Active surveillance of avian influenza virus (AIV) in wetlands and lakes is important for exploring the gene pool in wild birds. Through active surveillance from 2015 through 2019, 10,900 samples from wild birds in central China were collected, and 89 AIVs were isolated, including 2 subtypes of highly pathogenic AIV and 12 of low-pathogenic AIV; H9N2 and H6Ny were the dominant subtypes. Phylogenetic analysis of the isolates demonstrated that extensive intersubtype reassortments and frequent intercontinental gene exchange occurred in AIVs. AIV gene segments persistently circulated in several migration seasons, but interseasonal persistence of the whole genome was rare. The whole genomes of one H6N6 and polymerase basic 2 (PB2), polymerase acidic (PA), hemagglutinin (HA), neuraminidase (NA), M, and nonstructural (NS) genes of one H9N2 virus were found to be of poultry origin, suggesting a spillover of AIVs from poultry to wild birds. Importantly, one H9N2 virus only bound to human-type receptor, and one H1N1, four H6, and seven H9N2 viruses possessed dual receptor-binding capacity. Nineteen of 20 representative viruses tested could replicate in the lungs of mice without preadaptation, which poses a clear threat of infection in humans. Together, our study highlights the need for intensive AIV surveillance. IMPORTANCE Influenza virus surveillance in wild birds plays an important role in the early recognition and control of the virus. However, the AIV gene pool in wild birds in central China along the East Asian-Australasian flyway has not been well studied. Here, we conducted a 5-year AIV active surveillance in this region. Our data revealed the long-term circulation and prevalence of AIVs in wild birds in central China, and we observed that intercontinental gene exchange of AIVs is more frequent and continuous than previously thought. Spillover events from poultry to wild bird were observed in H6 and H9 viruses. In addition, in 20 representative viruses, 12 viruses could bind human-type receptors, and 19 viruses could replicate in mice without preadaption. Our work highlights the potential threat of wild bird AIVs to public health.

written and rigorous analyses are presented. A few points listed below need clarification and/or correction. Comments: 1. The pathogenicity characterization of the viruses is limited. Some controls are missing (especially the PBS control in Fig 6) and no statistics is done. Moreover, the viruses were recovered from mice at day 3 post infection but because there are no further time points, it is unknown whether they can actively replicate and grow in mammals. Standard viruses could have been used as controls to better conclude the experiments. Some precautions should be taken when concluding the results and the limitation of this experiment clearly mentioned. How to explain the H10N7 virus showed avian receptor binding specificity only but was recovered in mice in nasal and lungs? 2. There is no data regarding recent migratory seasons post 2019. I understand those data may not be processed yet in the area. Brief surveillance reports are published regularly and could be used here to discuss the data. Can the authors put into perspective their results with other available surveillance data in mainland China (H Bo, ...D-Y Wang, 2021 for example) and Europe/North America? Other specific comments: 3. Fig 1: can the authors indicate the number of total samples for each migratory season. A legend for the x axis is needed in panel C. This manuscript describes a five-year active surveillance in wild birds in Central China, along the East Asian-Australasian flyway, from 2015 to 2019. Twelve HA-NA subtype combinations were detected. Phylogenetic analysis revealed intercontinental transmission, continuous reassortment events. The manuscript provides valuable information on the genetic and biological properties of AIV from wild birds in China from 2015 to 2019. These data are important to understand the evolution, intercontinental spread and zoonotic potential of wild-bird AIV. The following comments should be addressed: Major comments:

Fig 6 and
1.it is not clear why this number of samples was collected. How the authors determined the sample size for their systemic study? The variable number of samples for each season may be the cause for the variable prevalence rate and subsequently the distribution of different subtypes per season. The authors should explain the reasons for these variations. 2.The assumption that a virus has zoonotic potential based on binding affinity to small glycan can be misleading. Replication kinetics of selected viruses (e.g. with high replication efficiency in mice) in human cells should be done. 3.Line 383: How did the author distinguish the droppings of free-range poultry from wild birds and identify the bird species?
Minor comments: 1. Line 85: More details on the EA flyway (e.g. no. of birds, destinations, time of migration, most common species, etc.) should be added in the introduction section to highlight the importance of this study. 2. Lines 153-159: it would be helpful to discuss these results in the discussion section. 3. Line 278: "..total of 20 representative strains", these AIV should be marked in the phylogenetic tree (at least in the supplementary figures).

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Thank you for submitting your paper to Microbiology Spectrum. We agree with you that it's better to test the viral titers of the organs at more time points and use standard viruses as control to get more meaningful and rigorous conclusions. We'll improve the experimental design in the future, and we have discussed the limitations in line 393-397 in the revised manuscript.

Reviewer(s)' Comments to Author
The α -2,3-linked receptors can be expressed in the lower respiratory tract and lungs of both human and mice, so some avian influenza viruses can replicate in the lungs of mammals but lack infectivity (Nature. 2006 Mar 23;440(7083):435-6.). The sialic acid receptor spectrum in mice is slightly different from that in humans. α -2,3 and α -2,6-linked sialic acid receptors are proved to be expressed both in the basal and connective tissue of the nasal of mice (Vet Res Commun. 2009 Dec;33(8):895-903.), which could explain the H10N7 virus showed avian receptor binding specificity only but was recovered in mice in nasal and lungs.  sampling is limited. Therefore, it is hard to guarantee that enough samples are collected in every sampling activity, and we just try to get as many fresh samples as we can. This is the reason why the sample size is variable among seasons. However, we sampled monthly in the migration seasons to avoid the sampling bias.

Q2:
The assumption that a virus has zoonotic potential based on binding affinity to small glycan can be misleading. Replication kinetics of selected viruses (e.g. with high replication efficiency in mice) in human cells should be done.
A2: Yes, we agree with you. Evaluating a virus has zoonotic potential based on binding affinity to small glycan can be misleading. So we tested the replication ability of the viruses in mice. Our data showed that most viruses (19/20) can replicate in mice without prior adaptation, and we think the data in mice infection are more powerful than replication kinetics of viruses in human cells.
Q3: How did the author distinguish the droppings of free-range poultry from wild birds and identify the bird species?
A3: The sampling sites is in the nature reserve, where free-range poultry is hard to access. And the shape of most fecal samples collected by us were quite different from poultry (as shown in the picture below).  Table 2.